A. Field of the Invention
The invention relates to the field of molecular biology; in particular, to immunogenic compositions and recombinant VMP-like genes useful for treatment and diagnosis of Lyme disease. Also included are methods for the determination of virulence factors in Lyme disease.
B. Description of Related Art
Lyme disease is a bacterial infection caused by pathogenic spirochetes of the genus Borrelia. The infection can occur in humans, dogs, deer, mice and other animals, and is transmitted by arthropod vectors, most notably ticks of the genus Ixodes. Borrelia burgdorferi, the most common cause of Lyme disease in North America, was first cultured in 1982. B. garinii and B. afzelii are the most common infectious agents of Lyme disease in Europe, and another species, B. japonicum, has been described in Japan. These organisms are closely related and cause similar manifestations with multiple stages: an expanding rash at the site of the tick bite (erythema migrans); fever, lymphadenopathy, fatigue, and malaise; effects of disseminated infection, including carditis, meningoradiculitis, and polyarthritis; and chronic manifestations including arthritis and neurologic disorders.
Lyme disease is often difficult to diagnose because of shared manifestations with other disorders, and it can also be refractory to treatment during late stages of the disease. It is most common in areas such as suburban regions of upstate New York and Connecticut, where large populations of deer and white-footed mice serve as the principal mammalian hosts and reservoirs of infection. Approximately 20,000 cases of Lyme disease in humans are reported per year in the United States, and it is also a significant veterinary problem due to a high infection rate of dogs and other domestic animals in endemic regions.
The pathogenic Borrelia that cause Lyme disease are able to persist for years in patients or animals despite the presence of an active immune response. Antigenic variation is a mechanism by which members of the genus Borrelia may be able to evade the host immune response (Zhang, 1997; Wang 2003). Antigenic variation has been defined as changes in the structure or expression of antigenic proteins that occurs during infection at a frequency greater than the usual mutation rate (Borst and Geaves, 1987; Robertson and Meyer, 1992; Seifert and So, 1988).
Relapsing fever is another disease caused by pathogenic Borrelia. It has both epidemic and endemic forms. The epidemic form is caused by B. recurrentis and is transmitted between humans by lice. It was a major source of morbidity and mortality during World War I, but has been rare since then due largely to public health measures. Endemic relapsing fever is an epizootic infection caused by several Borrelia species, including B. hermsii. It occurs sporadically among hunters, spelunkers, and others who come in contact with infected soft-bodied ticks of the genus Ornithidorus. Relapsing fever is characterized by two or more episodes or “relapses” of high bacteremia (up to 108/ml). The first wave of infection is caused by Borreliae expressing a certain Variable Major Protein (VMP) on their surface (e.g. Vmp21). The gene encoding this VMP is located at a promoter site in the expression plasmid, whereas over 24 nonexpressed copies of different VMP genes are present on the so-called silent plasmid. When the host develops antibodies against the expressed VMP, the organisms of that serotype are destroyed and the patient improves. However, a small proportion of organisms have undergone antigenic switching to a different serotype. Nonreciprocal recombination occurs between the expression plasmid and the silent plasmid, resulting in the insertion of a different VMP gene in the expression site (e.g., Vmp7). The organisms expressing Vmp7 are not affected by the anti-Vmp21 antibodies, and therefore multiply in the host and cause a second episode of the disease. Up to five of these 3-5 day episodes can occur, separated by 1-2 week intervals.
Such well-demarcated episodes of infection do not occur during Lyme disease, and fewer organisms are present in the blood at any stage. However, there are reasons to suspect that similar mechanisms of antigenic variation may occur in B. afzelii and other Lyme disease Borreliae such as B. garinii and B. burgdorferi. The infection, if untreated, commonly persists for months to years despite the occurrence of host antibody and cellular responses; this observation indicates effective evasion of the immune response. Lyme disease may be disabling (particularly in its chronic form), and thus there is a need for effective therapeutic and prophylactic treatment.
Genetic loci analogous to the VMP antigenic variation system have been detected in North American and European Lyme disease Borrelia by Southern hybridization and PCR analysis (Iyer et al., 2000; Wang et al, 2001). In addition, sequences from fragments of vls (VMP-like sequence) silent cassettes have been reported for the Borrelia burgdorferi strains 297 and N40, and the Borrelia garinii strains Ip90 and A87S (Kawabata et al., 1998; Liang and Philipp, 1999; Wang et al., 2001), (S. Feng and S. W. Barthold, unpublished data). VMP-like sequences of B. burgdorferi have been described and patented in U.S. Pat. No. 6,437,116.
Open reading frames in a B. burgdorferi plasmid that encode hypothetical proteins resembling the VMP proteins of relapsing fever organisms have been identified (Zhang et al., 1997). The inventors have found that the presence of the plasmid containing these VMP-like sequences in B. burgdorferi clones correlates strongly with infectivity (Zhang et al., 1997; Purser and Norris, 2000; Labandeira-Ray et al., 2001). Thus it is likely that the proteins encoded by the VMP-like sequences are important in immunoprotection and pathogenesis. Proteins encoded by the VMP-like sequences of B. burgdorferi may provide protection when used either alone or in combination with other antigens. They may also be useful for immunodiagnosis.
Greater than 90% of Lyme disease patients beyond the erythema migrans stage from North America and Europe express antibodies against VlsE (Lawrenz et al, 1999; Liang et al., 1999; Liang et al., 2000). In addition, mice infected experimentally with Borrelia afzelli and Borrelia garinii strains express anti-VlsE antibodies (Liang et al., 2000). Finally, a protein product of ˜35 kDa expressed by Borrelia garinii Ip90 reacts with antibodies against IR6, a peptide corresponding to invariant region 6 of the VlsE cassette region (Liang et al., 1999a). Portions of several vls silent cassettes from Borrelia garinii strain A87S have been published (Wang et al., 2001). Further, several amino acid sequences of Borrelia garinii Ip90 have been previously characterized by Liang et al. (1999a).
There is a commercial demand for vaccines and diagnostic kits for Lyme disease, both for human and veterinary use. Several companies have active research and development programs in these areas.